Process control for increased robustness of genetic assays

ABSTRACT

The present disclosure describes a method of processing a biological sample. The method includes contacting the sample with a pH indicator that changes color in response to changes in pH. The presence of a pH indicator such as cresol red provides the benefit of improved visualization of the sample, verification that the sample is properly frozen and thawed, and increased precision in pH adjustments during the assay, among other benefits.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of and priority to U.S. Provisional Patent Application Ser. No. 62/054,725, filed Sep. 24, 2014, the contents of which are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The invention generally relates to compositions and methods for processing a biological sample. More specifically, it relates to compositions and methods for analyzing a sample in a preimplantation genetic screening assay.

BACKGROUND

Preimplantation genetic screening (PGS) is a procedure in which one or more cells are biopsied from an embryo and analyzed to determine the karyotype of the embryo. The biopsy typically is performed by an embryologist, who places the cell(s) in a tube with 2-3 ul of liquid before freezing and delivering to a laboratory for testing. The laboratory receives the frozen liquid containing the biopsied cell(s), and performs a lysis procedure to liberate the nucleic acid. The lysis can be performed in several different ways, but the most common technique uses an alkaline solution, optionally in the presence of elevated temperature, to lyse the cell(s) and potentially denature and/or shear the nucleic acid for downstream analysis.

By profiling embryos for certain genetic disorders, a prospective parent may select embryos that lack the disorder, have an increased chance of successful pregnancy, have a lower predisposition to cancer, and a variety of other considerations. Preimplantation genetic diagnosis is used to select desired oocytes or embryos from a group of oocytes or fertilized embryos, some of which may be at risk, rather than assaying for a specific disease or condition. Preimplantation genetic screening can also be used to select embryos that have a euploid chromosome complement (22 pairs of autosomes and 2 sex chromosomes), or that lack finer-scale genomic rearrangements including translocations or sub-chromosomal gains or losses, thereby increasing the chance that the embryo will successfully implant, develop to term, and result in a healthy live birth, and decreasing the potential desire for selective pregnancy termination.

Preimplantation genetic screening may involve removing multiple eggs from a donor, fertilizing them to produce embryos, analyzing the embryos, and selecting an embryo that meets certain desired characteristics. To analyze a preimplanted embryo or blastocyst, only a small amount of cellular material can generally be biopsied. Because the embryo or blastocyst may have only a few dozen cells altogether, a PGS assay may involve biopsying only a single cell. Generally, the cell or cells are biopsied and analyzed to determine the karyotype of the embryo.

SUMMARY

The present disclosure provides compositions and related methods for processing a cellular sample. Specifically, methods of the invention involve utilizing an optical indicator in order to determine whether a liquid sample comprising cells is frozen prior to shipment for laboratory testing, and for monitoring and ensuring the integrity of the process in the laboratory that lyses the cells to free their nucleic acid for downstream analysis. Moreover, it has been discovered that certain indicators are also useful in stabilizing the sample as well as monitoring pH.

In one embodiment, a cellular sample is obtained by biopsy and placed in an appropriate buffer or collection storage fluid. The indicator, described more specifically below, is added and the sample is frozen. Alternately, a cellular sample can be obtained by biopsy and placed into an appropriate storage or collection fluid that already contains the indicator. A color change in the indicator is used to determine that the sample is frozen and is sufficient for delivery to a laboratory. The laboratory may be on the same premises that obtains and freezes the sample or may be remote (i.e., the frozen sample can simply be stored prior to delivery to laboratory personnel for testing). Alternatively, a change in electrical conductivity in the sample is used to monitor the indicator.

A particularly useful mode of practicing the invention is in the context of preimplantation genetic screening in which the quality and position of the frozen sample is critical prior to processing for genetic analysis. Biopsied embryonic material is placed in a collection tube with a collection buffer. An indicator is then added to the collection tube. Any indicator that is useful in determining whether the sample has been prepared properly may be used. However, a particularly useful indicator in the context of PGS is cresol red, or another triarylmethane dye. Cresol red is an example of a halochromic chromophore that changes color within the visible spectrum in response to changes in pH. The presence of cresol red aids in visualizing the small aliquot of liquid used in PGS, thereby allowing for improved monitoring of liquid placement and freezing, as well as monitoring fine pH adjustments. After the initial collection of sample and the addition of cresol red, the sample is often optionally frozen and shipped to another laboratory for further analysis. Proper freezing and thawing of the sample are necessary for maintaining the integrity of the cellular sample. Cresol red has been discovered to change color in response to freezing, and so its presence in the tube is useful for alerting a clinician handling the sample whether it has been properly frozen or thawed.

After thawing, the sample may be lysed by adding an alkaline lysis solution to the tube. The clinician performing the method can monitor the liquid for the appropriate color change and adjust the pH by titrating with an acid or base, as needed, to achieve the proper range for the lysis step. After lysis, the pH is neutralized by adding a neutralization solution and again monitoring for an appropriate color change, which signals that the sample has achieved the proper range. The pH can again be fine-tuned as necessary to ensure the appropriate neutral pH for subsequent downstream analysis.

The presence of cresol red or another pH indicator in the liquid mixture allows a clinician to ensure that: a) the liquid is in the bottom of the tube before freezing; b) the liquid is frozen before the tubes are shipped by the embryologist; c) the liquid is frozen when received by the laboratory; d) a volume of alkaline solution sufficient to increase pH is added to the tube; and e) a volume of acid sufficient to neutralize pH is added to the tube.

In alternative embodiments of the method, the pH indicator can be added at any step. Because a pH indicator such as cresol red provides multiple benefits at various steps throughout the method (i.e., visualizing the liquid before freezing, ensuring proper freezing and thawing, and monitoring subtle pH changes), it is beneficial to add it early in the procedure. However, those of skill in the art will understand that some assays may not require early addition of the pH indicator, such as assays that do not involve freezing, for example. Also, some assays that have other limitations, such as where the addition of pH indicator would interfere with an early step in the protocol, would also benefit from a later addition of the indicator.

After each step performed in the laboratory, the color change of the pH indicator can be monitored by eye. Alternatively it can be monitored automatically by a system including a camera controlled by computer software capable of determining the precise color of the solution, and therefore the precise pH. The computer software can flag any samples that are not at the correct pH for intervention to ensure that the solution is brought to the correct pH before downstream analytical steps are performed. If a sample has an incorrect pH, it can be adjusted by adding an appropriate amount of acid or base, either by automated means or manually by the clinician.

The method can be performed using cresol red, any other triarylmethane dye, or any other pH indicator that changes color in response to changes in pH, as long as the pH indicator is compatible with downstream analytical steps. For example, a pH indicator that is known to interfere with the activity of Taq polymerase would not be useful for an assay that required Taq-based PCR after lysis and neutralization.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the steps of a method for processing a biological sample.

FIGS. 2A and 2B show a variety of pH indicators and their characteristic color ranges.

FIG. 3 shows a system configured to identify color changes in a halochromic chromophore using a camera.

DETAILED DESCRIPTION

The present disclosure describes methods of processing a biological sample. The method includes contacting the sample with an indicator that changes color in response to changes in pH. The presence of a pH indicator, such as cresol red, provides the benefit of improved visualization of the sample, verification that the sample is properly frozen and thawed, and increased precision in pH adjustments during the assay, among other benefits.

The method is particularly useful for preimplantation genetic screening (PGS). PGS assays generally involve small samples in small volumes of liquid, which must be frozen and thawed prior to analysis. Additionally, PGS protocols require various pH adjustments that must be precise in order to maintain the efficacy of the assay. The addition of a pH indicator such as cresol red to the liquid mixture allows a clinician to ensure that: a) the liquid is in the bottom of the tube before freezing; b) the liquid is frozen before the tubes are shipped by the embryologist; c) the liquid is frozen when received by the laboratory; d) a volume of alkaline solution sufficient to increase pH is added to the tube; and e) a volume of acid sufficient to neutralize pH is added to the tube.

While the methods described herein are particularly useful for PGS analysis, they can also be used for other assays of biological materials. In addition to assaying an embryonic sample, the methods can be used to analyze a buccal sample, a blood sample, an amniotic fluid sample, a cervical sample, an ocular sample, or any other sample comprising cells or cellular materials including nucleic acid. The methods described herein can be adapted to any assay known in the art that requires one or more steps involving a change in pH of the sample or reaction mixture.

Although a preferred embodiment of the invention involves the use of cresol red, the method can be performed using another triarylmethane dye. Triarylmethane dyes are synthetic organic compounds containing triphenylmethane backbones, and presenting intense colors in the visual spectrum. Many react to changes in pH, and are therefore useful as pH indicators. Any other pH indicator can be used with methods of the invention, as long as it changes color or otherwise reacts in response to changes in pH, and it is compatible with downstream analytical steps. For example, a pH indicator that is known to interfere with the activity of Taq polymerase would not be useful for an assay that involved Taq-based PCR downstream of the lysis and neutralization steps.

FIG. 1 is a flowchart 100 depicting a series of steps in a preferred embodiment of the invention. The flowchart 100 shows steps for processing a biological sample by methods of the present disclosure. In step 110, a sample comprising cells is obtained from a subject. The sample can be comprised of one or more cells biopsied from an embryo or oocyte of a subject. After the sample is collected it is contacted with a collection medium in step 120. The collection medium can be a buffer, an alcohol, a preservative medium, or any other such medium that would be known to a person skilled in the art. Often for PGS analysis, the volume of collection medium is very small. It can be, for example, 1 μl or less. The volume can also be about 2 μl, 3 μl, 4 μl, 5 μl, 10 μl, 20 μl, 50 μl, 100 μl, or more, depending on the needs of the assay.

After step 120, the sample may be frozen and shipped to another lab to perform the subsequent steps of the procedure. Alternatively, all steps can be performed in a single lab, obviating the need to freeze and thaw the sample. In embodiments of the invention that involve freezing the collection sample, the collection sample is thawed prior to performing step 130. In step 130, the cells in the collection sample are lysed by the addition of an alkaline lysis solution to the mixture. In other embodiments of the method, the cells are lysed using other techniques, such as mechanical shearing, osmotic lysing, chemical denaturing, centrifuging, sonicating, freeze-thawing, or other techniques known in the art.

When lysis is performed using an alkaline solution, such as in step 130, optionally the solution can be exposed to elevated temperatures to further catalyze the lysis process. In step 130 the cell or cells in the mixture are lysed and nucleic acids therein may also be denatured or sheared, preparing them for downstream analysis.

Following step 130, the solution is neutralized in step 140 by adding a neutralization solution. Generally the neutralization solution will be acidic, to neutralize the alkaline lysis solution. The lysis step 130 and the neutralizing step 140 both require precision to achieve proper pH levels. Imprecision in the pipetted volume of either the lysis reagent or the neutralizing reagent will result in a suboptimal pH. If the pH after step 140 is not neutral, for example, the downstream steps may be impeded, which may lead to assay failure.

For that reason, among others, a pH indicator is added in step 150. A pH indicator such as cresol red is useful with methods of the invention. Cresol red is a halochromic chromophore that changes color within the visible spectrum in response to changes in pH. It appears yellow below pH 7.2, red until pH 8.8, and violet above pH 8.8. The color change of the pH indicator can be monitored by eye, or it can be monitored automatically by a system including a camera controlled by computer software capable of determining the precise color of the solution, and therefore the precise pH. The computer software can flag any samples that are not at the correct pH, and a clinician can intervene to ensure that the solution is brought to the optimal pH before downstream analytical steps are performed. That intervention can also be automated, whereby the computer system triggers a pH adjustment in response to identifying an incorrect pH. The pH adjustments, whether performed manually by a lab technician or automatically by a computer system, can be achieved by adding an appropriate amount of acid or base as necessary.

In various embodiments of FIG. 1, step 150 can occur immediately downstream of any of steps 110-140. Methods of the invention include the step of contacting the sample with an indicator 150 immediately after step 110, step 120, step 130, or step 140. In other embodiments, step 150 is performed simultaneously with steps 120, 130, 140, or 150.

The addition of a pH indicator such as cresol red has advantages at various stages of the method. The presence of cresol red or another compatible pH indicator helps monitor the sample throughout a procedure that requires multiple fine pH adjustments. Further, as discussed above, the sample is often frozen and shipped to another laboratory for further analysis after the initial collection of sample. That means that proper freezing and thawing of the sample are necessary for maintaining the integrity of the cellular sample. It has been discovered that cresol red changes color in response to freezing and thawing, and so its presence in the sample mixture at steps 120, 130, and 140 help to verify that the sample has been properly handled. Additionally, cresol red allows easier visualization of small amounts of liquid in the sample tube. Therefore, the presence of cresol red is especially useful in PGS and other similar assays, where the volume is small and the clinician must ensure that the sample is at the bottom of the tube prior to freezing.

In addition to cresol red, other pH indicators that are useful in other embodiments of the disclosed invention are gentian violet (Methyl violet 10B), malachite green, thymol blue, methyl yellow, bromophenol blue, congo red, methyl orange, bromocresol green, methyl red, azolitmin, bromocresol purple, bromothymol blue, phenol red, neutral red, naphtholphthalein, cresolphthalein, phenolphthalein, thymolphthalein, alizarine yellow R, and any other common laboratory pH indicator known in the art.

FIGS. 2A-B show a list of several pH indicators that can be used with the invention, along with the characteristic color changes for each indicator at different pH levels. As shown in FIGS. 2A-B, several indicators have overlapping ranges for most levels of pH. That means that for a given assay requiring a specific pH, numerous pH indicators could be used. For the PGS assay described above, indicators such as neutral red and phenol red have similar ranges as cresol red, and therefore could be substituted. For assays involving different pH requirements, a pH indicator can be selected that has a suitable range that would allow an observer to differentiate between subtle pH variances as needed.

In addition to those listed in FIGS. 2A-B, other pH indicators include anthocyanins, litmus, and hydrangea flowers. Additionally, fluorescent dyes can be used as direct pH indicators, as fluorescence is a function of pH. Nucleic acid reporter molecules, which denature at pH extremes and renature or otherwise change structural formation at neutral pH, can also be used. These can be coupled with flourophore/quencher pair, a FRET pair, or mixed with intercalating dye for structural readout. Protein reporter molecules, which change conformation as a function of pH, can be used in a similar manner as can pH sensitive fluorescent proteins (see, e.g., Kneen, 1998, “Green fluorescent protein as a noninvasive intracellular pH indicator,” Biophys J. 74(3):1591-99; Miesenböck, 1998, “Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins,” Nature 394(6689):192-95; and Llopis, 1998, “Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins,” Proc. Natl. Acad. Sci. U.S.A. 95(12):6803-08).

Additionally, pH can be measured electronically using a pH meter. Electronic pH meters are available, such as the Hach HQ440D Benchtop Meter, available from Hach Company (Loveland, Col.). Also available are miniaturized pH meters such as Ion Torrent or ion-sensitive field-effect transistors, which detect ion concentration changes in a solution and respond with a changed current through a transistor. As discussed above, in all cases, the selected pH measurement technique should be compatible with downstream analytical steps. Some halochromic chromophores, such as bromophenol blue, for example, are known to interfere with the normal activity of Taq polymerase, and so would not be an optimal choice if a Taq-based PCR were to be performed on the nucleic acid after lysis and neutralization.

The disclosure additionally provides a composition of matter, the composition comprising a sample comprising one or more cells, a collection medium, an alkaline lysis solution, a neutralization solution, and a pH indicator. The composition provides a reaction mixture for PGS analysis that is more stable and that is easier to adjust for desired pH levels than compositions of the prior art. The composition can comprise a sample that has been biopsied from an embryo. Alternatively the sample can be a buccal sample, a blood sample, an amniotic fluid sample, a cervical sample, an ocular sample, or any other sample comprising cells or cellular materials including nucleic acid. The collection medium can be a buffer, an alcohol, or a preservative medium, or any other similar medium known in the art. The neutralization solution comprises any solution that neutralizes the pH of the lysis solution. Therefore, when the lysis solution is alkaline, the neutralization solution is acidic. The pH indicator can be cresol red, or any other pH indicator listed in FIGS. 2A-B or otherwise known in the art. The composition can be produced in conjunction with the methods described herein.

As discussed above, the color changes of the pH indicator can be observed by an automated mechanism such as a camera connected to a computer. As one skilled in the art would recognize as necessary or best-suited for performance of the methods of the invention, computer system or machines of the invention include one or more processors (e.g., a central processing unit (CPU) a graphics processing unit (GPU) or both), a main memory and a static memory, which communicate with each other via a bus.

In an exemplary embodiment shown in FIG. 3, system 200 can include a camera 201 with data acquisition module 205 to obtain sequence read data. Camera 201 may optionally include or be operably coupled to its own, e.g., dedicated, camera computer 233 (including an input/output mechanism 237, one or more of processor 241 and memory 245). Additionally or alternatively, camera 201 may be operably coupled to a server 213 or computer 249 (e.g., laptop, desktop, or tablet) via network 209. Computer 249 includes one or more processor 259 and memory 263 as well as an input/output mechanism 254. Where methods of the invention employ a client/server architecture, steps of methods of the invention may be performed using server 213, which includes one or more of processor 221 and memory 229, capable of obtaining data, instructions, etc., or providing results via interface module 225 or providing results as a file 217. Server 213 may be engaged over network 209 through computer 249 or terminal 267, or server 213 may be directly connected to terminal 267, including one or more processor 275 and memory 279, as well as input/output mechanism 271.

System 200 or machines according to the invention may further include, for any of I/O 249, 237, or 271 a video display unit (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)). Computer systems or machines according to the invention can also include an alphanumeric input device (e.g., a keyboard), a cursor control device (e.g., a mouse), a disk drive unit, a signal generation device (e.g., a speaker), a touchscreen, an accelerometer, a microphone, a cellular radio frequency antenna, and a network interface device, which can be, for example, a network interface card (NIC), Wi-Fi card, or cellular modem.

Memory 263, 245, 279, or 229 according to the invention can include a machine-readable medium on which is stored one or more sets of instructions (e.g., software) embodying any one or more of the methodologies or functions described herein. The software may also reside, completely or at least partially, within the main memory and/or within the processor during execution thereof by the computer system, the main memory and the processor also constituting machine-readable media.

The software may further be transmitted or received over a network via the network interface device.

While the machine-readable medium can in an exemplary embodiment be a single medium, the term “machine-readable medium” should be taken to include a single medium or multiple media (e.g., a centralized or distributed database, and/or associated caches and servers) that store the one or more sets of instructions. The term “machine-readable medium” shall also be taken to include any medium that is capable of storing, encoding or carrying a set of instructions for execution by the machine and that cause the machine to perform any one or more of the methodologies of the present invention. The term “machine-readable medium” shall accordingly be taken to include, but not be limited to, solid-state memories (e.g., subscriber identity module (SIM) card, secure digital card (SD card), micro SD card, or solid-state drive (SSD)), optical and magnetic media, and any other tangible storage media.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

EQUIVALENTS

Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof. 

What is claimed is:
 1. A method for processing a biological sample, the method comprising: obtaining a sample comprising at least one cell biopsied from an embryo in a solution of 2-3 μl of liquid; contacting the sample with an indicator dye comprising cresol red that provides a visual indication that the sample is frozen and within a specific, pre-defined pH range; exposing the sample to a freezing temperature; determining based on the visual indication that the sample is frozen; and performing a preimplantation genetic screening procedure comprising: using a camera controlled by computer software to evaluate a color of the sample; confirming with the computer software that the color indicates that the sample remains frozen; contacting the sample with an alkaline lysis solution followed by contacting the sample with an acid to achieve a neutral pH; using the camera and the computer software to evaluate a color of the sample and confirm that the sample has a neutral pH; and analyzing a karyotype of the at least one biopsied cell in the sample.
 2. The method of claim 1, further comprising: assessing said indicator in order to determine if the pH range is appropriate; and delivering the sample to a laboratory if said indicator indicates that the sample is frozen.
 3. The method of claim 1, further comprising the step of assessing said indicator in order to determine a position of said cells in a vial containing the sample.
 4. The method of claim 1, wherein the alkaline lysis solution contains said indicator; and wherein the acid contains said indicator.
 5. The method of claim 2, wherein said assessing step is performed by observing a color change in the sample.
 6. The method of claim 5, wherein color change is monitored by automatic means, wherein the automatic means comprises a camera.
 7. The method of claim 2, wherein said assessing step is performed by monitoring electrical conductivity in the sample. 